Project Summary Robert syndrome (RBS) is a severe genetic disorder characterized by phocomelia (flipper-like appendages), microcephaly, cleft palate, syndactyly, intellectual disabilities, seizures, and abnormalities in the heart and urinary, genital, and alimentary tract tissues. Importantly, mutation of ESCO2 is the single and only requirement necessary to elicit this multi- tissue and systemic suite of phenotypes. It follows that elucidating the mechanisms through which Esco2 functions will significantly advance our understanding of pathway integrations required for human development - from the physical to the cognitive. Esco2 (yeast Eco1) is an acetyltransferase that activates the DNA tethering complex - cohesin. Esco2/Eco1 and cohesin are both essential for proper chromosome segregation. In turn, the prevailing model of RBS (ESCO2 mutated) is that birth defects arise from mitotic failure (massive aneuploidy) and subsequent loss (apoptosis) of proliferative stem cells and developing tissues. Previously, we posited an alternate model in which RBS arises from transcription dysregulation. A collaborative effort indeed discovered that Esco2 and cohesin co-regulate the transcription of genes critical for bone growth. Thus, testing new and alternate models of Esco2 function will significantly advance our understanding of human development. Whether predicated on mitotic failure/apoptosis or transcriptional dysregulation, all models of RBS focus on Esco2 acetylation of the cohesin subunit Smc3. The multi-faceted nature of RBS, however, suggests that additional signaling pathways are at play. Here, we report new evidence that Eco1 (yeast homolog of Esco2) may target substrates beyond cohesins. Characterization of these novel Esco2/Eco1 substrates, and testing their roles specifically in development, provides the first novel approach to understanding RBS, and numerous other developmental maladies, to emerge in recent years. The Specific Aims of this proposal are interdependent and contain conceptually distinct genetic approaches from which to identify novel substrates of Eco1 acetylation reactions. The technical aspects of the two screens (an unbiased genome-wide cohesin-bypass screen and a genome-wide conditional dosage lethality screen) are complete ? with genetic interactors in hand. In each aim, we validate the candidates, assess the acetylation state of the gene products and dependency on Eco1, and test the importance of substrate acetylation in yeast. We then translate findings into zebrafish embryos from which we can directly test for roles in development and also RBS cells to test for changes in targeted substrate modifications.